Respiratory tract pathogens such as the human parainfluenza virus and SARS coronavirus effectively infect human airways by exploiting a diverse set of cell surface glycans and glycoprotein receptors encountered in airway cell types. In case of adeno-associated viruses (AAV), serotypes 1, 5, 6 and more recently, AAV9 have been shown to efficiently transduce airways in vitro and in vivo, albeit with striking species-specific and serotype-specific differences. The goal of this proposal is to elucidate molecular and cellular determinants of airway tropism in AAV serotypes. To achieve such, we have devised a comprehensive approach that hinges on molecular manipulation of AAV capsids through mutagenesis, biochemical reagents for identification of glycans and co-receptors that mediate AAV airway cell entry and relevant in vitro models of the human respiratory tract. The strategies described herein will (a) define AAV capsid structural elements at the amino acid level that determine airway tropism, and (b) enable identification of cell surface components including glycans and integrin subunits that dictate AAV tropism for different airway cell types. The proposed studies will help provide a comprehensive picture of the mechanisms underlying AAV airway cell entry as well as provide insight into species-specific differences in AAV airway tropism. If successful, this knowledge may facilitate improvements in AAV vectors as well as in the design of preclinical/clinical studies focused on gene therapy of airway diseases such as cystic fibrosis. Public Health Relevance: The goal of this proposal is to understand the molecular mechanisms exploited by AAV in infecting human airways. To achieve such, we will use a comprehensive approach including molecular virology techniques, novel biochemical reagents and a panel of different human airway cell types. If successful, this knowledge may facilitate improvements in AAV vectors as well as in the design of preclinical/clinical studies focused on gene therapy of airway diseases such as cystic fibrosis.